U.S. patent application number 15/041743 was filed with the patent office on 2016-08-18 for pharmaceutical compositions comprising perillyl alcohol derivatives.
The applicant listed for this patent is NEONC TECHNOLOGIES INC.. Invention is credited to Thomas Chen, Daniel Levin, Satish Pupalli.
Application Number | 20160237092 15/041743 |
Document ID | / |
Family ID | 56615134 |
Filed Date | 2016-08-18 |
United States Patent
Application |
20160237092 |
Kind Code |
A1 |
Chen; Thomas ; et
al. |
August 18, 2016 |
PHARMACEUTICAL COMPOSITIONS COMPRISING PERILLYL ALCOHOL
DERIVATIVES
Abstract
A pharmaceutical composition is provided which includes perillyl
alcohol conjugated with a therapeutic agent and further includes
and a hydrolyzable acylated aliphatic tail. A method of using the
pharmaceutical composition is also provided for treating a
condition or disease of a patient, e.g., cancer.
Inventors: |
Chen; Thomas; (La Canada,
CA) ; Levin; Daniel; (La Canada, CA) ;
Pupalli; Satish; (Rancho Cucamonga, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
NEONC TECHNOLOGIES INC. |
Los Angeles |
CA |
US |
|
|
Family ID: |
56615134 |
Appl. No.: |
15/041743 |
Filed: |
February 11, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62115396 |
Feb 12, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07D 231/12 20130101;
C07D 487/04 20130101; A61P 35/00 20180101 |
International
Class: |
C07D 487/04 20060101
C07D487/04 |
Claims
1. A compound of the Formula I; ##STR00048## wherein R is selected
from the group consisting of a C.sub.4 to C.sub.28 linear or
branched alkyl, linear or branched C.sub.4 to C.sub.28 alkeneyl and
a linear or branched C.sub.4 to C.sub.28 alkynyl group; or a
pharmaceutically acceptable salt thereof.
2. The compound according to claim 1 wherein R is a C.sub.4 to
C.sub.28 linear or branched alkeneyl containing 1, 2, 3, 4 or 5
double bonds or a linear or branched C.sub.4 to C.sub.28 alkynyl
group containing 1, 2, 3, 4 or 5 triple bonds.
3. The compound according to claim 1 of the Formula II;
##STR00049## or a pharmaceutically acceptable salt thereof.
4. A method for treating a disease in a mammal, comprising the step
of delivering to the mammal a therapeutically effective amount of
the compound according to claim 1, 2 or 3.
5. The method of claim 4, wherein the disease is cancer.
6. The method of claim 5, wherein the cancer is a tumor of the
nervous system.
7. The method of claim 6, wherein the tumor is a glioblastoma.
8. The method of claim 5, wherein the cancer is a skin cancer
selected from the group of melanoma, basal cell carcinoma, and
squamous cell carcinoma.
9. The method of claim 8, wherein the cancer is melanoma.
10. The method of claim 9, wherein the disease is a precancerous
skin lesion.
11. The method of claim 4, wherein the compound is administered by
inhalation, intranasally, orally, intravenously, topically,
transdermally, subcutaneously or intramuscularly.
12. The method of claim 11, wherein the composition is administered
topically.
13. A pharmaceutical composition comprising a compound according to
claim 1, 2 or 3.
14. A compound of the Formula III; ##STR00050## wherein R.sub.1 is
selected from a C.sub.4 to C.sub.28 linear or branched alkyl,
linear or branched C.sub.4 to C.sub.28 alkeneyl or a linear or
branched C.sub.4 to C.sub.28 alkynyl group; or a pharmaceutically
acceptable salt thereof.
15. The compound according to claim 14 wherein R.sub.1 is a C.sub.4
to C.sub.28 linear or branched alkeneyl containing 1, 2, 3, 4 or 5
double bonds or a linear or branched C.sub.4 to C.sub.28 alkynyl
group containing 1, 2, 3, 4 or 5 triple bonds.
16. The compound according to claim 14 of the Formula IV; or a
pharmaceutically acceptable salt thereof. ##STR00051##
17. A method for treating a disease in a mammal, comprising the
step of delivering to the mammal a therapeutically effective amount
of the compound according to claim 14, 15 or 16.
18. The method of claim 17, wherein the disease is cancer.
19. The method of claim 18, wherein the cancer is a tumor of the
nervous system.
20. The method of claim 19, wherein the tumor is a
glioblastoma.
21. The method of claim 18, wherein the cancer is a skin cancer
selected from the group of melanoma, basal cell carcinoma, and
squamous cell carcinoma.
22. The method of claim 21, wherein the cancer is melanoma.
23. The method of claim 22, wherein the disease is a precancerous
skin lesion.
24. The method of claim 14, wherein the compound is administered by
inhalation, intranasally, orally, intravenously, topically,
transdermally, subcutaneously or intramuscularly.
25. The method of claim 24, wherein the composition is administered
topically.
26. A pharmaceutical composition comprising a compound according to
claim 14, 15 or 16.
27. A process for making a compound of the Formula I ##STR00052##
comprising the steps of a) reacting Temozolamide with oxalyl
chloride in a halogenated solvent to give the isocyanate of
Temozolamide Formula A ##STR00053## b) reacting the isocyanate of
Temozolamide with perillyl alcohol in a halogenated solvent to
afford a compound of Formula B ##STR00054## c) reacting the
compound of Formula B with an acid chloride of the Formula C
##STR00055## wherein R is selected from the group consisting of a
C.sub.4 to C.sub.28 linear or branched alkyl, linear or branched
C.sub.4 to C.sub.28 alkeneyl containing 1, 2, 3, 4, or 5 double
bonds or a linear or branched C.sub.4 to C.sub.28 alkynyl group;
containing 1, 2, 3, 4 or 5 triple bonds in the presence of a base
in an ethereal solvent to afford a compound of the Formula I.
28. The process according to claim 27 wherein the halogenated
solvent is 1,2 dichloroethane.
29. The process according to claim 27 wherein the acid chloride is
linoleic acid chloride.
30. The process according to claim 27 wherein the base is NaH.
31. The process according to claim 27 wherein the ethereal solvent
is THF.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Patent
Application Ser. No. 62/115,396, filed on Feb. 12, 2015 which is
incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to monoterpene derivatives
containing a therapeutic agent as well as an aliphatic tail. The
present invention further relates to methods and compositions of
using the monoterpene derivatives to treat cancer.
BACKGROUND
[0003] Malignant gliomas, the most common form of central nervous
system (CNS) cancers, is currently considered essentially
incurable. Among the various malignant gliomas, anaplastic
astrocytomas (Grade III) and glioblastoma multiforme (GBM; Grade
IV) have an especially poor prognosis due to their aggressive
growth and resistance to currently available therapies. The present
standard of care for malignant gliomas consists of surgery,
ionizing radiation, and chemotherapy. Despite recent advances in
medicine, the past 50 years have not seen any significant
improvement in prognosis for malignant gliomas. Wen et al.
Malignant gliomas in adults. New England J Med. 359: 492-507, 2008.
Stupp et al. Radiotherapy plus concomitant and adjuvant
temozolomide for glioblastoma. New England J Med. 352: 987-996,
2005.
[0004] The poor response of tumors, including malignant gliomas, to
various types of chemotherapeutic agents are often due to intrinsic
drug resistance. Additionally, acquired resistance of initially
well-responding tumors and unwanted side effects are other problems
that frequently thwart long-term treatment using chemotherapeutic
agents. Hence, various analogues of chemotherapeutic agents have
been prepared in an effort to overcome these problems. The
analogues include novel therapeutic agents which are hybrid
molecules of at least two existing therapeutic agents. For example,
cisplatin has been conjugated with Pt-(II) complexes with cytotoxic
codrugs, or conjugated with bioactive shuttle components such as
porphyrins, bile acids, hormones, or modulators that expedite the
transmembrane transport or the drug accumulation within the cell.
(6-Aminomethylnicotinate) dichloridoplatinum(II) complexes
esterified with terpene alcohols were tested on a panel of human
tumor cell lines. The terpenyl moieties in these complexes appeared
to fulfill a transmembrane shuttle function and increased the rate
and extent of the uptake of these conjugates into various tumor
cell lines. Schobert et al. Monoterpenes as Drug Shuttles:
Cytotoxic (6-minomethylnicotinate) dichloridoplatinum(II) Complexes
with Potential to Overcome Cisplatin Resistance. J. Med. Chem.
2007, 50, 1288-1293.
[0005] Perillyl alcohol (POH), a naturally occurring monoterpene,
has been suggested to be an effective agent against a variety of
cancers, including CNS cancer, breast cancer, pancreatic cancer,
lung cancer, melanomas and colon cancer. Gould, M. Cancer
chemoprevention and therapy by monoterpenes. Environ Health
Perspect. 1997 June; 105 (Suppl 4): 977-979. Hybrid molecules
containing both perillyl alcohol and retinoids were prepared to
increase apoptosis-inducing activity. Das et al. Design and
synthesis of potential new apoptosis agents: hybrid compounds
containing perillyl alcohol and new constrained retinoids.
Tetrahedron Letters 2010, 51, 1462-1466.
[0006] Co-owned U.S. Patent Publication No. 20130203828 disclosed
various perillyl alcohol derivatives such as perillyl alcohol
carbamates. For example, the perillyl alcohol derivatives include
perillyl alcohol conjugated with a therapeutic agent such as
dimethyl celocoxib (DMC), temozolomide (TMZ) or rolipram.
[0007] There is still a need for perillyl alcohol derivatives with
improved properties for transdermal or topical application, as well
as use the derivatives in the treatment of cancers such as
malignant gliomas, skin cancers as well as precancerous skin
conditions.
SUMMARY OF THE INVENTION
[0008] In one aspect of the present application, a pharmaceutical
composition is provided. The composition comprises a compound
comprising perillyl alcohol conjugated with a therapeutic agent,
and an acylated aliphatic tail. The aliphatic tail can be derived
from fatty acids. In some embodiments, the aliphatic tail contains
4 to 28 carbon atoms. The aliphatic chain can be saturated or
unsaturated, branched or non-branched.
[0009] In one embodiment, the compound is a perillyl alcohol
carbamate, where the nitrogen of the carbamate group is acylated
with the aliphatic tail. In one embodiment, the therapeutic agent
is a chemotherapeutic agent. The chemotherapeutic agent includes,
but is not limited to, of a DNA alkylating agent, a topoisomerase
inhibitor, an endoplasmic reticulum stress inducing agent, a
platinum compound, an antimetabolite, an enzyme inhibitor, and a
receptor antagonist. In specific embodiments, the therapeutic agent
can be dimethyl celocoxib (DMC), temozolomide (TMZ), or
rolipram.
[0010] In one embodiment, the compound is
(3-Methyl-4-oxo-3,4-dihydro-imidazo[5,1-d][1,2,3,5]tetrazine-8-carbonyl)--
octadeca-9,12-dienoyl-carbamic acid
4-isopropenyl-cyclohex-1-enylmethyl ester. In another embodiment,
the compound is
(3-Methyl-4-oxo-3,4-dihydro-imidazo[5,1-d][1,2,3,5]tetrazine-8-carbonyl)--
hexadecanoic acid 4-isopropenyl-cyclohex-1-enylmethyl ester.
[0011] In another aspect, there is provided compounds of the
Formula I;
##STR00001##
wherein R is selected from a C.sub.4 to C.sub.28 linear or branched
alkyl, C.sub.4 to C.sub.28 alkeneyl or a C.sub.4 to C.sub.28
alkynyl group; or a pharmaceutically acceptable salt thereof.
[0012] In another aspect there is provided a compound according to
Formula I wherein R is a C.sub.4 to C.sub.28 linear or branched
alkeneyl containing 1, 2, 3, 4 or 5 double bonds.
[0013] In another aspect there is provided a compound according to
Formula I wherein R is a C.sub.4 to C.sub.28 linear or branched
alkynyl containing 1, 2, 3, 4 or 5 triple bonds.
In another aspect a compound of the Formula II;
##STR00002##
or a pharmaceutically acceptable salt thereof is provided. In
another aspect, a compound of the Formula III is provided;
##STR00003##
wherein R.sub.1 is selected from a C.sub.4 to C.sub.28 linear or
branched alkyl, linear or branched C.sub.4 to C.sub.28 alkeneyl or
a linear or branched C.sub.4 to C.sub.28 alkynyl group; or a
pharmaceutically acceptable salt thereof. In another aspect there
is provided a compound according to Formula III wherein R.sub.1 is
a C.sub.4 to C.sub.28 linear or branched alkeneyl containing 1, 2,
3, 4 or 5 double bonds.
[0014] In another aspect there is provided a compound according to
Formula III wherein R.sub.1 is a C.sub.4 to C.sub.28 linear or
branched alkynyl containing 1, 2, 3, 4 or 5 triple bonds.
In another aspect a compound of the Formula IV; or a
pharmaceutically acceptable salt thereof is provided.
##STR00004##
[0015] In another aspect, the present invention provides a method
for treating a disease in a mammal. The method comprises delivering
to the mammal a therapeutically effective amount of a
pharmaceutical composition described herein. In one embodiment, the
disease is cancer. For example, the cancer is a tumor of the
nervous system, e.g., glioblastoma. The cancer can also be a skin
cancer, such as melanoma, basal cell carcinoma, and squamous cell
carcinoma. In another embodiment, the disease is a precancerous
skin lesion. The composition can be administered by inhalation,
intranasally, orally, intravenously, topically, transdermally,
subcutaneously or intramuscularly.
In another aspect a process for making a compound of the Formula
I
##STR00005##
comprising the steps of
[0016] a) reacting Temozolamide with oxalyl chloride in a
halogenated solvent, preferably 1,2 dichloroethane, to give the
isocyanate of Temozolamide Formula A
##STR00006##
[0017] b) reacting the isocyanate of Temozolamide with perillyl
alcohol in a halogenated solvent to afford a compound of Formula
B
##STR00007##
[0018] c) reacting the compound of Formula B with an acid chloride,
preferably linoleic acid chloride, of the Formula C
##STR00008##
wherein R is selected from the group consisting of a C.sub.4 to
C.sub.28 linear or branched alkyl, linear or branched C.sub.4 to
C.sub.28 alkeneyl containing 1, 2, 3, 4, or 5 double bonds or a
linear or branched C.sub.4 to C.sub.28 alkynyl group; containing 1,
2, 3, 4 or 5 triple bonds in the presence of a base, preferably
NaH, in an ethereal solvent, preferably THF, to afford a compound
of the Formula I is provided.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
[0020] FIG. 1 is a plot showing in vitro efficacy of temozolomide
(TMZ), TMZ-POH, and TMZ-POH-linoleate (TPL) in the treatment of
MGMT negative human melanoma cells A2058.
[0021] FIG. 2 is a plot showing in vitro efficacy of temozolomide
(TMZ), TMZ-POH, and TPL in the treatment of MGMT positive human
melanoma cells A375.
[0022] FIG. 3A is an image of a tumor formed on the skin of a nude
mouse due to subcutaneously injected melanoma cells.
[0023] FIG. 3B is an image of the tumor depicted in FIG. 3A after
being treated with TPL for 14 days.
[0024] FIGS. 4A-4C are example micrographs of the tissue stained
for visualization of CD31 marker expression obtained from a tumor
formed on the skin of a nude mouse due to subcutaneously injected
melanoma cells, where the mouse had been treated by a vehicle for
14 days.
[0025] FIGS. 4D-4F are example micrographs of the tissue stained
for visualization of CD31 marker expression obtained from a nodule
formed on the skin of a nude mouse due to subcutaneously injected
melanoma cells, where the mouse had been treated by TPL for 14
days.
[0026] FIG. 5 is a plot comparing the average area representing
CD31 marker expression in a nodule formed on the skin of a nude
mouse due to subcutaneously injected melanoma cells which had been
treated with TPL for 14 days with that of a control mouse treated
with a vehicle.
DETAILED DESCRIPTION
[0027] In one aspect, the present invention provides for
derivatives of monoterpene (or sesquiterpene) comprising a
monoterpene (or sesquiterpene) conjugated with a therapeutic agent.
The monoterpene (or sesquiterpene) derivative can further include
an acylated aliphatic tail. In some embodiments, the aliphatic tail
contains 4 to 28 carbon atoms. As an example, the monoterpene (or
sesquiterpene) can be perillyl alcohol (POH). The therapeutic agent
may be covalently linked with the monoterpene (or sesquiterpene)
through carbamate, ester, ether bonds, or any other suitable
chemical functional groups. For example, the monoterpene derivative
can be perillyl alcohol carbamate of a therapeutic agent wherein
the nitrogen of the carbamate group is acylated with the aliphatic
tail. The therapeutic agent can be a chemotherapeutic agent, such
as a DNA alkylating agent, a topoisomerase inhibitor, an
endoplasmic reticulum stress inducing agent, a platinum compound,
an antimetabolite, an enzyme inhibitor, or a receptor antagonist.
In particular embodiments, the therapeutic agent can be dimethyl
celocoxib (DMC), temozolomide (TMZ), or rolipram. The molar ratio
of the monoterpene (or sesquiterpene) to the therapeutic agent in
the monoterpene (or sesquiterpene) conjugate may be 1:1, 1:2, 1:3,
1:4, 2:1, 3:1, 4:1, or any other suitable molar ratios. When the
monoterpene (or sesquiterpene) and the therapeutic agent are
conjugated through a carbamate bond, the therapeutic agent may be
any agent bearing at least one carboxylic acid functional group, or
any agent bearing at least one amine functional group. The
aliphatic chain can be saturated or unsaturated, straight chain or
branched-chain.
[0028] As used herein, monoterpenes include terpenes that consist
of two isoprene units. Monoterpenes may be linear (acyclic) or
contain rings. Derivatives of monoterpenoids are also encompassed
by the present invention. Monoterpenoids may be produced by
biochemical modifications such as oxidation or rearrangement of
monoterpenes. Examples of monoterpenes and monoterpenoids include,
perillyl alcohol (S(-)) and (R(+)), ocimene, myrcene, geraniol,
citral, citronellol, citronellal, linalool, pinene, terpineol,
terpinen, limonene, terpinenes, phellandrenes, terpinolene,
terpinen-4-ol (or tea tree oil), pinene, terpineol, terpinen; the
terpenoids such as p-cymene which is derived from monocyclic
terpenes such as menthol, thymol and carvacrol; bicyclic
monoterpenoids such as camphor, borneol and eucalyptol.
[0029] Monoterpenes may be distinguished by the structure of a
carbon skeleton and may be grouped into acyclic monoterpenes (e.g.,
myrcene, (Z)- and (E)-ocimene, linalool, geraniol, nerol,
citronellol, myrcenol, geranial, citral a, neral, citral b,
citronellal, etc.), monocyclic monoterpenes (e.g., limonene,
terpinene, phellandrene, terpinolene, menthol, carveol, etc.),
bicyclic monoterpenes (e.g., pinene, myrtenol, myrtenal, verbanol,
verbanon, pinocarveol, carene, sabinene, camphene, thujene, etc.)
and tricyclic monoterpenes (e.g. tricyclene). See Encyclopedia of
Chemical Technology, Fourth Edition, Volume 23, page 834-835.
[0030] Sesquiterpenes of the present invention include terpenes
that consist of three isoprene units. Sesquiterpenes may be linear
(acyclic) or contain rings. Derivatives of sesquiterpenoids are
also encompassed by the present invention. Sesquiterpenoids may be
produced by biochemical modifications such as oxidation or
rearrangement of sesquiterpenes. Examples of sesquiterpenes include
farnesol, farnesal, farnesylic acid and nerolidol.
[0031] The derivatives of monoterpene (or sesquiterpene) include,
but are not limited to, carbamates, esters, ethers, alcohols and
aldehydes of the monoterpene (or sesquiterpene). Monoterpene (or
sesquiterpene) alcohols may be derivatized to carbamates, esters,
ethers, aldehydes or acids.
[0032] Carbamate refers to a class of chemical compounds sharing
the functional group
##STR00009##
based on a carbonyl group flanked by an oxygen and a nitrogen. The
R groups on the nitrogen and the oxygen may form a ring.
R.sup.1--OH may be a monoterpene, e.g., POH. In some embodiments,
one of R.sup.2 and R.sup.3 may be a therapeutic agent, while the
other of R.sup.2 and R.sup.3 is an acylated aliphatic chain having
4-28 carbons.
[0033] Carbamates may be synthesized by reacting isocyanate and
alcohol, or by reacting chloroformate with amine. Carbamates may be
synthesized by reactions making use of phosgene or phosgene
equivalents. For example, carbamates may be synthesized by reacting
phosgene gas, diphosgene or a solid phosgene precursor such as
triphosgene with two amines or an amine and an alcohol. Carbamates
(also known as urethanes) can also be made from reaction of a urea
intermediate with an alcohol. Dimethyl carbonate and diphenyl
carbonate are also used for making carbamates. Alternatively,
carbamates may be synthesized through the reaction of alcohol
and/or amine precursors with an ester-substituted diaryl carbonate,
such as bismethylsalicylcarbonate (BMSC). U.S. Patent Publication
No. 20100113819.
[0034] Carbamates may be synthesized by the following approach:
##STR00010##
Monoterpenes or sesquiterpines (e.g POH) are reacted with phosgene
in the presence of a first base such and an aromatic solvent such
as toluene to form the corresponding chloroformate. The
chloroformates are then reacted with a therapeutic agent having an
NH.sub.2 group (e.g DMC) optionally in the presence of a second
base to afford the carbamate. The carbamates can then be reacted
with an acid chloride of a C.sub.4 to C.sub.28 linear or branched
alkyl carboxylic acid, a C.sub.4 to C.sub.28 alkeneyl carboxylic
acid having 1-5 double bonds or a C.sub.4 to C.sub.28 alkynyl
carboxylic acid having 1-5 triple bonds; optionally in the presence
of a third base. Suitable reaction solvents include, but are not
limited to, tetrahydrofuran, dichloromethane, dichloroethane,
acetone, and diisopropyl ether. The reaction may be performed at a
temperature ranging from about -70.degree. C. to about 80.degree.
C., or from about -65.degree. C. to about 50.degree. C. The molar
ratio of perillyl chloroformate (or the chloroformate of a
monoterpene or sesquiterpene) to the substrate R.sub.1--NH.sub.2
(wherein R.sub.1--NH.sub.2 is a therapeutic agent) may range from
about 1:1 to about 2:1, from about 1:1 to about 1.5:1, from about
2:1 to about 1:1, or from about 1.05:1 to about 1.1:1. Suitable
first, second and third bases include, but are not limited to,
organic bases, such as triethylamine, N,N'-diisopropylethylamine,
butyl lithium, and potassium-t-butoxide and inorganic bases such as
sodium or potassium carbonate, KOH, NaOH and NaH.
[0035] Alternatively, carbamates may be synthesized by the
following approach:
##STR00011##
R.sub.2C(O)NH.sub.2 (where R.sub.2C(O)NH.sub.2 is a therapeutic
agent e.g TMZ) is reacted with oxalyl chloride to produce the
isocyanate (R.sub.2--N.dbd.C.dbd.O) followed by reaction with a
monoterpene or sesquiterpene (e.g POH) to afford the carbamate,
optionally in the presence of a first base. The carbamates can then
be reacted with an acid chloride of a C.sub.4 to C.sub.28 linear or
branched alkyl carboxylic acid, a C.sub.4 to C.sub.28 alkeneyl
carboxylic acid having 1-5 double bonds or a C.sub.4 to C.sub.28
alkynyl carboxylic acid having 1-5 triple bonds; optionally in the
presence of a second base. Suitable reaction solvents include, but
are not limited to, dichloromethane, dichloroethane, toluene,
diisopropyl ether, and tetrahydrofuran. The reaction may be
performed at a temperature ranging from about 25.degree. C. to
about 110.degree. C., or from about 30.degree. C. to about
80.degree. C., or about 50.degree. C. The molar ratio of perillyl
alcohol (or monoterpene or sesquiterpene) to the substrate
R.sub.2--N.dbd.C.dbd.O may range from about 1:1 to about 2:1, from
about 1:1 to about 1.5:1, from about 2:1 to about 1:1, or from
about 1.05:1 to about 1.1:1. Suitable first, second and third bases
include, but are not limited to, organic bases, such as
triethylamine, N,N'-diisopropylethylamine, butyl lithium, and
potassium-t-butoxide or inorganic bases such as but not limited to
potassium or sodium carbonate, NaOH, KOH, and NaH.
[0036] Esters of the monoterpene (or sesquiterpene) alcohols of the
present invention can be derived from an inorganic acid or an
organic acid. Inorganic acids include, but are not limited to,
phosphoric acid, sulfuric acid, and nitric acid. Organic acids
include, but are not limited to, carboxylic acid such as benzoic
acid, fatty acid, acetic acid and propionic acid, and any
therapeutic agent bearing at least one carboxylic acid functional
group Examples of esters of monoterpene (or sesquiterpene) alcohols
include, but are not limited to, carboxylic acid esters (such as
benzoate esters, fatty acid esters (e.g., palmitate ester,
linoleate ester, stearate ester, butyryl ester and oleate ester),
acetates, propionates (or propanoates), and formates), phosphates,
sulfates, and carbamates (e.g., N,N-dimethylaminocarbonyl).
[0037] A specific example of a monoterpene that may be used in the
present invention is perillyl alcohol. The derivatives of perillyl
alcohol include perillyl alcohol carbamates, perillyl alcohol
esters, perillic aldehydes, dihydroperillic acid, perillic acid,
perillic aldehyde derivatives, dihydroperillic acid esters and
perillic acid esters.
[0038] In certain embodiments, a POH carbamate is synthesized by a
process comprising the step of reacting a first reactant of
perillyl chloroformate with a second reactant such as dimethyl
celocoxib (DMC), temozolomide (TMZ) and rolipram. The reaction may
be carried out in the presence of tetrahydrofuran and a base such
as n-butyl lithium. Perillyl chloroformate may be made by reacting
POH with phosgene. For example, POH conjugated with temozolomide
through a carbamate bond may be synthesized by reacting
temozolomide with oxalyl chloride followed by reaction with
perillyl alcohol. The reaction may be carried out in the presence
of 1,2-dichloroethane.
[0039] As described herein, the monoterpene derivative, such as POH
carbamate conjugated with another therapeutic agent, can be further
reacted with a fatty acid to form a tri-conjugate structure
containing an acylated aliphatic tail (which is the fatty acid with
the terminal --OH of the carboxyl group removed). The fatty acid
can be unsaturated, such as Caprylic acid, Capric acid, Lauric
acid, Myristic acid, Palmitic acid, Stearic acid, Arachidic acid,
Behenic acid, Lignoceric acid, Cerotic acid; or saturated, such as
Myristoleic acid, Palmitoleic acid, Sapienic acid, Oleic acid,
Elaidic acid, Vaccenic acid, Linoleic acid, Linoelaidic acid,
.alpha.-Linolenic acid, Arachidonic acid, Eicosapentaenoic acid,
Erucic acid, Docosahexaenoic acid. For example, when the
therapeutic agent is TMZ, the fatty acid can be linoleic acid to
form
(3-Methyl-4-oxo-3,4-dihydro-imidazo[5,1-d][1,2,3,5]tetrazine-8-carbonyl)--
octadeca-9,12-dienoyl-carbamic acid
4-isopropenyl-cyclohex-1-enylmethyl ester, or it can be palmitic
acid to form
(3-Methyl-4-oxo-3,4-dihydro-imidazo[5,1-d][1,2,3,5]tetrazine-8-carbonyl)--
hexadecanoic acid 4-isopropenyl-cyclohex-1-enylmethyl ester.
Halides of the fatty acid (e.g., acyl bromide, acyl chloride of
these fatty acids), or anhydrides of the fatty acid can also be
used. For example, a POH carbamate that is a conjugate with a
therapeutic agent can be further reacted with a fatty acid (or a
fatty acid halide or anhydride) such that the hydrogen of the
--NH-- group in the carbamate linker group is substituted with the
acylated aliphatic tail of the fatty acid.
Except where stated otherwise, the following definitions apply
throughout the present specification and claims. These definitions
apply regardless of whether a term is used by itself or in
combination with other terms. For example, the definition of
"alkyl" applies not only to alkyl groups per se, but also to the
alkyl portions of alkoxy, alkylamino, alkylthio or alkylcarbonyl
groups etc. Furthermore all ranges described for a chemical group,
for example "from 1 to 13 carbon atoms" or "C.sub.1-C.sub.6 alkyl"
include all combinations and sub-combinations of ranges and
specific numbers of carbon atoms therein.
[0040] As described herein, "Alkyl" means a straight (linear) chain
or branched chain aliphatic hydrocarbon group having from 4 to 28
carbon atoms in the chain. Preferred alkyl groups have from 10 to
20 carbon atoms in the chain. More preferred alkyl groups have from
14 to 20 carbon atoms in the chain. Non limiting examples of
suitable alkyl groups includeisopropyl, sec-butyl, n-butyl, and
t-butyl.
[0041] As described herein "Alkenyl" means a straight (linear)
chain or branched chain aliphatic hydrocarbon group having 1 to 8,
preferably 1 to 5, more preferably 1 to 3, carbon-carbon double
bonds and having from 4 to 28 carbon atoms in the chain. Preferred
alkenyl groups have from 10 to 20 carbon atoms in the chain. More
preferred alkenyl groups have from 14 to 20 carbon atoms in the
chain. Non limiting examples of suitable alkenyl groups include
isopropenyl, n-butenyl, 1-hexenyl and 3-methylbut-2-enyl.
[0042] As described herein "Alkynyl" means a straight (linear)
chain or branched chain aliphatic hydrocarbon group having at 1 to
5 carbon-carbon triple bonds and having from 4 to 28 carbon atoms
in the chain. Preferred alkynyl groups have from 4 to 12 carbon
atoms in the chain. More preferred alkynyl groups have from 4 to 6
carbon atoms in the chain. Examples of suitable 2-propynyl and
2-butynyl.
[0043] According to the present invention, the therapeutic agents
that may be conjugated with monoterpene (or sesquiterpene) include,
but are not limited to, chemotherapeutic agents, therapeutic agents
for treatment of CNS disorders (including, without limitation,
primary degenerative neurological disorders such as Alzheimer's,
Parkinson's, multiple sclerosis, Attention-Deficit Hyperactivity
Disorder or ADHD, psychological disorders, psychosis and
depression), immunotherapeutic agents, angiogenesis inhibitors, and
anti-hypertensive agents. Anti-cancer agents that may be conjugated
with monoterpene or sesquiterpene can have one or more of the
following effects on cancer cells or the subject: cell death;
decreased cell proliferation; decreased numbers of cells;
inhibition of cell growth; apoptosis; necrosis; mitotic
catastrophe; cell cycle arrest; decreased cell size; decreased cell
division; decreased cell survival; decreased cell metabolism;
markers of cell damage or cytotoxicity; indirect indicators of cell
damage or cytotoxicity such as tumor shrinkage; improved survival
of a subject; or disappearance of markers associated with
undesirable, unwanted, or aberrant cell proliferation. U.S. Patent
Publication No. 20080275057.
[0044] Also encompassed by the present invention is admixtures
and/or coformulations of a monoterpene (or sesquiterpene) and at
least one therapeutic agent.
[0045] Chemotherapeutic agents include, but are not limited to, DNA
alkylating agents, topoisomerase inhibitors, endoplasmic reticulum
stress inducing agents, a platinum compound, an antimetabolite,
vincalkaloids, taxanes, epothilones, enzyme inhibitors, receptor
antagonists, tyrosine kinase inhibitors, boron radiosensitizers
(i.e. velcade), and chemotherapeutic combination therapies.
[0046] Non-limiting examples of DNA alkylating agents are nitrogen
mustards, such as Cyclophosphamide (Ifosfamide, Trofosfamide),
Chlorambucil (Melphalan, Prednimustine), Bendamustine, Uramustine
and Estramustine; nitrosoureas, such as Carmustine (BCNU),
Lomustine (Semustine), Fotemustine, Nimustine, Ranimustine and
Streptozocin; alkyl sulfonates, such as Busulfan (Mannosulfan,
Treosulfan); Aziridines, such as Carboquone, Triaziquone,
Triethylenemelamine; Hydrazines (Procarbazine); Triazenes such as
Dacarbazine and Temozolomide (TMZ); Altretamine and
Mitobronitol.
[0047] Non-limiting examples of Topoisomerase I inhibitors include
Campothecin derivatives including SN-38, APC, NPC, campothecin,
topotecan, exatecan mesylate, 9-nitrocamptothecin,
9-aminocamptothecin, lurtotecan, rubitecan, silatecan, gimatecan,
diflomotecan, extatecan, BN-80927, DX-8951f, and MAG-CPT as
decribed in Pommier Y. (2006) Nat. Rev. Cancer 6(10):789-802 and
U.S. Patent Publication No. 200510250854; Protoberberine alkaloids
and derivatives thereof including berberrubine and coralyne as
described in Li et al. (2000) Biochemistry 39(24):7107-7116 and
Gatto et al. (1996) Cancer Res. 15(12):2795-2800; Phenanthroline
derivatives including Benzo[i]phenanthridine, Nitidine, and
fagaronine as described in Makhey et al. (2003) Bioorg. Med. Chem.
11 (8): 1809-1820; Terbenzimidazole and derivatives thereof as
described in Xu (1998) Biochemistry 37(10):3558-3566; and
Anthracycline derivatives including Doxorubicin, Daunorubicin, and
Mitoxantrone as described in Foglesong et al. (1992) Cancer
Chemother. Pharmacol. 30(2):123-]25, Crow et al. (1994) J. Med.
Chem. 37(19):31913194, and Crespi et al. (1986) Biochem. Biophys.
Res. Commun. 136(2):521-8. Topoisomerase II inhibitors include, but
are not limited to Etoposide and Teniposide. Dual topoisomerase I
and II inhibitors include, but are not limited to, Saintopin and
other Naphthecenediones, DACA and other Acridine-4-Carboxamindes,
Intoplicine and other Benzopyridoindoles, TAS-I03 and other
7H-indeno[2,1-c]Quinoline-7-ones, Pyrazoloacridine, XR 11576 and
other Benzophenazines, XR 5944 and other Dimeric compounds,
7-oxo-7H-dibenz[f,ij]Isoquinolines and
7-oxo-7H-benzo[e]pyrimidines, and Anthracenyl-amino Acid Conjugates
as described in Denny and Baguley (2003) Curr. Top. Med. Chem.
3(3):339-353. Some agents inhibit Topoisomerase II and have DNA
intercalation activity such as, but not limited to, Anthracyclines
(Aclarubicin, Daunorubicin, Doxorubicin, Epirubicin, Idarubicin,
Amrubicin, Pirarubicin, Valrubicin, Zorubicin) and Antracenediones
(Mitoxantrone and Pixantrone).
[0048] Examples of endoplasmic reticulum stress inducing agents
include, but are not limited to, dimethyl-celecoxib (DMC),
nelfinavir, celecoxib, and boron radiosensitizers (i.e. velcade
(Bortezomib)).
[0049] Platinum based compounds are a subclass of DNA alkylating
agents. Non-limiting examples of such agents include Cisplatin,
Nedaplatin, Oxaliplatin, Triplatin tetranitrate, Satraplatin,
Aroplatin, Lobaplatin, and JM-216. (see McKeage et al. (1997) J.
Clin. Oncol. 201:1232-1237 and in general, CHEMOTHERAPY FOR
GYNECOLOGICAL NEOPLASM, CURRENT THERAPY AND NOVEL APPROACHES, in
the Series Basic and Clinical Oncology, Angioli et al. Eds.,
2004).
[0050] "FOLFOX" is an abbreviation for a type of combination
therapy that is used to treat colorectal cancer. It includes 5-FU,
oxaliplatin and leucovorin. Information regarding this treatment is
available on the National Cancer Institute's web site, cancer.gov,
last accessed on Jan. 16, 2008.
[0051] "FOLFOX/BV" is an abbreviation for a type of combination
therapy that is used to treat colorectal cancer. This therapy
includes 5-FU, oxaliplatin, leucovorin and Bevacizumab.
Furthennore, "XELOX/BV" is another combination therapy used to
treat colorectal cancer, which includes the prodrug to 5-FU, known
as Capecitabine (Xeloda) in combination with oxaliplatin and
bevacizumab. Information regarding these treatments are available
on the National Cancer Institute's web site, cancer.gov or from 23
the National Comprehensive Cancer Network's web site, nccn.org,
last accessed on May 27, 2008.
[0052] Non-limiting examples of antimetabolite agents include Folic
acid based, i.e. dihydrofolate reductase inhibitors, such as
Aminopterin, Methotrexate and Pemetrexed; thymidylate synthase
inhibitors, such as Raltitrexed, Pemetrexed; Purine based, i.e. an
adenosine deaminase inhibitor, such as Pentostatin, a thiopurine,
such as Thioguanine and Mercaptopurine, a
halogenated/ribonucleotide reductase inhibitor, such as Cladribine,
Clofarabine, Fludarabine, or a guanine/guanosine: thiopurine, such
as Thioguanine; or Pyrimidine based, i.e. cytosine/cytidine:
hypomethylating agent, such as Azacitidine and Decitabine, a DNA
polymerase inhibitor, such as Cytarabine, a ribonucleotide
reductase inhibitor, such as Gemcitabine, or a thymine/thymidine:
thymidylate synthase inhibitor, such as a Fluorouracil (5-FU).
Equivalents to 5-FU include prodrugs, analogs and derivative
thereof such as 5'-deoxy-5-fluorouridine (doxifluroidine),
1-tetrahydrofuranyl-5-fluorouracil (ftorafur), Capecitabine
(Xeloda), S-I (MBMS-247616, consisting of tegafur and two
modulators, a 5-chloro-2,4-dihydroxypyridine and potassium
oxonate), ralititrexed (tomudex), nolatrexed (Thymitaq, AG337),
LY231514 and ZD9331, as described for example in Papamicheal (1999)
The Oncologist 4:478-487.
[0053] Examples of vincalkaloids, include, but are not limited to
Vinblastine, Vincristine, Vinflunine, Vindesine and
Vinorelbine.
[0054] Examples of taxanes include, but are not limited to
docetaxel, Larotaxel, Ortataxel, Paclitaxel and Tesetaxel. An
example of an epothilone is iabepilone.
[0055] Examples of enzyme inhibitors include, but are not limited
to farnesyltransferase inhibitors (Tipifarnib); CDK inhibitor
(Alvocidib, Seliciclib); proteasome inhibitor (Bortezomib);
phosphodiesterase inhibitor (Anagrelide; rolipram); IMP
dehydrogenase inhibitor (Tiazofurine); and lipoxygenase inhibitor
(Masoprocol). Examples of receptor antagonists include, but are not
limited to ERA (Atrasentan); retinoid X receptor (Bexarotene); and
a sex steroid (Testolactone).
[0056] Examples of tyrosine kinase inhibitors include, but are not
limited to inhibitors to ErbB: HER1/EGFR (Erlotinib, Gefitinib,
Lapatinib, Vandetanib, Sunitinib, Neratinib); HER2/neu (Lapatinib,
Neratinib); RTK class III: C-kit (Axitinib, Sunitinib, Sorafenib),
FLT3 (Lestaurtinib), PDGFR (Axitinib, Sunitinib, Sorafenib); and
VEGFR (Vandetanib, Semaxanib, Cediranib, Axitinib, Sorafenib);
bcr-abl (Imatinib, Nilotinib, Dasatinib); Src (Bosutinib) and Janus
kinase 2 (Lestaurtinib).
[0057] "Lapatinib" (Tykerb.RTM.) is an dual EGFR and erbB-2
inhibitor. Lapatinib has been investigated as an anticancer
monotherapy, as well as in combination with trastuzumab,
capecitabine, letrozole, paclitaxel and FOLFIRI (irinotecan,
5-fluorouracil and leucovorin), in a number of clinical trials. It
is currently in phase III testing for the oral treatment of
metastatic breast, head and neck, lung, gastric, renal and bladder
cancer.
[0058] A chemical equivalent of lapatinib is a small molecule or
compound that is a tyrosine kinase inhibitor (TKI) or alternatively
a HER-1 inhibitor or a HER-2 inhibitor. Several TKIs have been
found to have effective antitumor activity and have been approved
or are in clinical trials. Examples of such include, but are not
limited to, Zactima (ZD6474), Iressa (gefitinib), imatinib mesylate
(STI571; Gleevec), erlotinib (OSI-1774; Tarceva), canertinib (CI
1033), semaxinib (SU5416), vatalanib (PTK787/ZK222584), sorafenib
(BAY 43-9006), sutent (SUI 1248) and lefltmomide (SU101).
[0059] PTK/ZK is a tyrosine kinase inhibitor with broad specificity
that targets all VEGF receptors (VEGFR), the platelet-derived
growth factor (PDGF) receptor, c-KIT and c-Fms. Drevs (2003) Idrugs
6(8):787-794. PTK/ZK is a targeted drug that blocks angiogenesis
and lymphangiogenesis by inhibiting the activity of all known
receptors that bind VEGF including VEGFR-I (Flt-1), VEGFR-2
(KDR/Flk-1) and VEGFR-3 (Flt-4). The chemical names of PTK/ZK are
1-[4-Chloroanilino]-4-[4-pyridylmethyl] phthalazine Succinate or
1-Phthalazinamine,
N-(4-chlorophenyl)-4-(4-pyridinylmethyl)-butanedioate (1:1).
Synonyms and analogs of PTK/TK are known as Vatalanib, CGP79787D,
PTK787/ZK 222584, CGP-79787, DE-00268, PTK-787, PTK787A, VEGFR-TK
inhibitor, ZK 222584 and ZK.
[0060] Chemotherapeutic agents that can be conjugated with
monoterpene or sesquiterpene may also include amsacrine,
Trabectedin, retinoids (Alitretinoin, Tretinoin), Arsenic trioxide,
asparagine depleter Asparaginase/Pegaspargase), Celecoxib,
Demecolcine, Elesclomol, Elsamitrucin, Etoglucid, Lonidamine,
Lucanthone, Mitoguazone, Mitotane, Oblimersen, Temsirolimus, and
Vorinostat.
[0061] The monoterpene or sesquiterpene derivative may be
conjugated with angiogenesis inhibitors. Examples of angiogenesis
inhibitors include, but are not limited to, angiostatin, angiozyme,
antithrombin III, AG3340, VEGF inhibitors, batimastat, bevacizumab
(avastin), BMS-275291, CAI, 2C3, HuMV833 Canstatin, Captopril,
carboxyamidotriazole, cartilage derived inhibitor (CDI), CC-5013,
6-O-(chloroacetyl-carbonyl)-fumagillol, COL-3, combretastatin,
combretastatin A4 Phosphate, Dalteparin, EMD 121974 (Cilengitide),
endostatin, erlotinib, gefitinib (Iressa), genistein, halofuginone
hydrobromide, Id1, Id3, IM862, imatinib mesylate, IMC-IC11
Inducible protein 10, interferon-alpha, interleukin 12, lavendustin
A, LY317615 or AE-941, marimastat, mspin, medroxpregesterone
acetate, Meth-1, Meth-2, 2-methoxyestradiol (2-ME), neovastat,
oteopontin cleaved product, PEX, pigment epithelium growth factor
(PEGF), platelet factor 4, prolactin fragment, proliferin-related
protein (PRP), PTK787/ZK 222584, ZD6474, recombinant human platelet
factor 4 (rPF4), restin, squalamine, SU5416, SU6668, SU11248
suramin, Taxol, Tecogalan, thalidomide, thrombospondin, TNP-470,
troponin-1, vasostatin, VEG1, VEGF-Trap, and ZD6474.
[0062] Non-limiting examples of angiogenesis inhibitors also
include, tyrosine kinase inhibitors, such as inhibitors of the
tyrosine kinase receptors Flt-1 (VEGFR1) and Flk-1/KDR (VEGFR2),
inhibitors of epidermal-derived, fibroblast-derived, or platelet
derived growth factors, MMP (matrix metalloprotease) inhibitors,
integrin blockers, pentosan polysulfate, angiotensin II
antagonists, cyclooxygenase inhibitors (including non-steroidal
anti-inflammatory drugs (NSAIDs) such as aspirin and ibuprofen, as
well as selective cyclooxygenase-2 inhibitors such as celecoxib and
rofecoxib), and steroidal anti-inflammatories (such as
corticosteroids, mineralocorticoids, dexamethasone, prednisone,
prednisolone, methylpred, betamethasone).
[0063] Other therapeutic agents that modulate or inhibit
angiogenesis and may also be conjugated with monoterpene or
sesquiterpene include agents that modulate or inhibit the
coagulation and fibrinolysis systems, including, but not limited
to, heparin, low molecular weight heparins and carboxypeptidase U
inhibitors (also known as inhibitors of active thrombin activatable
fibrinolysis inhibitor [TAFIa]). U.S. Patent Publication No.
20090328239. U.S. Pat. No. 7,638,549.
[0064] Non-limiting examples of the anti-hypertensive agents
include angiotensin converting enzyme inhibitors (e.g., captopril,
enalapril, delapril etc.), angiotensin II antagonists (e.g.,
candesartan cilexetil, candesartan, losartan (or Cozaar), losartan
potassium, eprosartan, valsartan (or Diovan), termisartan,
irbesartan, tasosartan, olmesartan, olmesartan medoxomil etc.),
calcium antagonists (e.g., manidipine, nifedipine, amlodipine (or
Amlodin), efonidipine, nicardipine etc.), diuretics, renin
inhibitor (e.g., aliskiren etc.), aldosterone antagonists (e.g.,
spironolactone, eplerenone etc.), beta-blockers (e.g., metoprolol
(or Toporol), atenolol, propranolol, carvedilol, pindolol etc.),
vasodilators (e.g., nitrate, soluble guanylate cyclase stimulator
or activator, prostacycline etc.), angiotensin vaccine, clonidine
and the like. U.S. Patent Publication No. 20100113780.
[0065] Other therapeutic agents that may be conjugated with
monoterpene (or sesquiterpene) include, but are not limited to,
Sertraline (Zoloft), Topiramate (Topamax), Duloxetine (Cymbalta),
Sumatriptan (Imitrex), Pregabalin (Lyrica), Lamotrigine (Lamictal),
Valaciclovir (Valtrex), Tamsulosin (Flomax), Zidovudine (Combivir),
Lamivudine (Combivir), Efavirenz (Sustiva), Abacavir (Epzicom),
Lopinavir (Kaletra), Pioglitazone (Actos), Desloratidine
(Clarinex), Cetirizine (Zyrtec), Pentoprazole (Protonix),
Lansoprazole (Prevacid), Rebeprazole (Aciphex), Moxifloxacin
(Avelox), Meloxicam (Mobic), Dorzolamide (Truspot), Diclofenac
(Voltaren), Enlapril (Vasotec), Montelukast (Singulair), Sildenafil
(Viagra), Carvedilol (Coreg), Ramipril (Delix).
[0066] Table 1 lists pharmaceutical agents that can be conjugated
with monoterpene (or sesquiterpene), including structure of the
pharmaceutical agent and the preferred derivative for
conjugation.
TABLE-US-00001 TABLE 1 Brand Generic Preferred Name Name Activity
Structure Derivative Zoloft Sertraline Depression ##STR00012##
Carbamate Topamax Topiramate Seizures ##STR00013## Carbamate
Cymbalta Duloxetine Depression ##STR00014## Carbamate Imitrex
Sumatriptan Migraine ##STR00015## Carbamate Lyrica Pregabalin
Neuropathic pain ##STR00016## Carbamate or Ester Lamictal
Lamotrigine Seizures ##STR00017## Carbamate Valtrex Valaciclovir
Herpes ##STR00018## Carbamate Tarceva Erlotinib Non-small cell lung
cancer ##STR00019## Carbamate Flomax Tamsulosin Benign prostatic
Cancer ##STR00020## Carbamate Gleevec Imatinib Leukemia
##STR00021## Carbamate Combivir Zidovudine HIV infection
##STR00022## Carbamate Combivir Lamivudine HIV infection
##STR00023## Carbonate Sustiva Efavirenz HIV infection ##STR00024##
Carbamate Epzicom Abacavir HIV infection ##STR00025## Carbamate
Kaletra Lopinavir HIV infection ##STR00026## Carbamate Actos
Pioglitazone Type-2 diabetes ##STR00027## Carbamate Clarinex
Desloratidine Allergic rhinitis ##STR00028## Carbamate Zyrtec
Cetirizine Allergic ##STR00029## Ester Protonix Pentoprazole
Gastrointestinal ##STR00030## Carbamate Prevacid Lansoprazole
Gastrointestinal ##STR00031## Carbamate Aciphex Rebeprazole
Gastrointestinal ##STR00032## Carbamate Diovan Valsartan
Hypertension ##STR00033## Carbamate Cozaar Losartan Hypertension
##STR00034## Carbamate Avelox Moxifloxacin Bacterial infection
##STR00035## Carbamate or Ester Mobic Meloxicam Osteoarthritis
##STR00036## Carbamate Truspot Dorzolamide Intraocular pressure
##STR00037## Carbamate Voltaren Diclofenac Osteoarthritis &
rheumatoid arthritis ##STR00038## Carbamate or Ester Vasotec
Enlapril Hypertension ##STR00039## Carbamate or Ester Singulair
Montelukast Asthma ##STR00040## Ester Amlodin Amlodipine
Hypertension ##STR00041## Carbamate Toporol Metoprolol Hypertension
##STR00042## Carbamate Viagra Sildenafil Erectile dysfunction
##STR00043## Carbamate Coreg Carvedilol Hypertension ##STR00044##
Carbamate Delix Ramipril Hypertension ##STR00045## Carbamate or
Ester Sinemet (Parcopa, Atamet) L-DOPA Neurological disorders
##STR00046##
[0067] The purity of the monoterpene (or sesquiterpene) derivatives
may be assayed by gas chromatography (GC) or high pressure liquid
chromatography (HPLC). Other techniques for assaying the purity of
monoterpene (or sesquiterpene) derivatives and for determining the
presence of impurities include, but are not limited to, nuclear
magnetic resonance (NMR) spectroscopy, mass spectrometry (MS),
GC-MS, infrared spectroscopy (IR), and thin layer chromatography
(TLC). Chiral purity can be assessed by chiral GC or measurement of
optical rotation.
[0068] The monoterpene (or sesquiterpene) derivatives may be
purified by methods such as crystallization, or by separating the
monoterpene (or sesquiterpene) derivative from impurities according
to the unique physicochemical properties (e.g., solubility or
polarity) of the derivative. Accordingly, the monoterpene (or
sesquiterpene) derivative can be separated from the monoterpene (or
sesquiterpene) by suitable separation techniques known in the art,
such as preparative chromatography, (fractional) distillation, or
(fractional) crystallization.
[0069] The invention also provides for methods of using the herein
described monoterpenes (or sesquiterpenes) derivatives to treat a
disease, such as cancer or other nervous system disorders. For
example, the cancer is a tumor of the nervous system, e.g.,
glioblastoma. The cancer can also be a skin cancer, such as
melanoma, basal cell carcinoma, or squamous cell carcinoma. The
disease can also be a precancerous skin lesion.
[0070] A monoterpenes (or sesquiterpenes) derivative may be
administered alone, or in combination with radiation, surgery or
additional chemotherapeutic agents. A monoterpene or sesquiterpene
derivative may also be co-administered with antiviral agents,
anti-inflammatory agents or antibiotics. The agents may be
administered concurrently or sequentially. A monoterpene (or
sesquiterpene) derivative can be administered before, during or
after the administration of the other active agent(s).
[0071] The monoterpene or sesquiterpene derivative may be used in
combination with radiation therapy. In one embodiment, the present
invention provides for a method of treating tumor cells, such as
malignant glioma cells, with radiation, where the cells are treated
with an effective amount of a monoterpene derivative, such as a
perillyl alcohol carbamate, and then exposed to radiation.
Monoterpene derivative treatment may be before, during and/or after
radiation. For example, the monoterpene or sesquiterpene derivative
may be administered continuously beginning one week prior to the
initiation of radiotherapy and continued for two weeks after the
completion of radiotherapy. U.S. Pat. Nos. 5,587,402 and
5,602,184.
[0072] In one embodiment, the present invention provides for a
method of treating tumor cells, such as malignant glioma cells,
with chemotherapy, where the cells are treated with an effective
amount of a monoterpene derivative, and then exposed to
chemotherapy. Monoterpene derivative treatment may be before,
during and/or after chemotherapy.
[0073] The monoterpene (or sesquiterpene) derivatives of the
present invention may be used for the treatment of nervous system
cancers, such as a malignant glioma (e.g., astrocytoma, anaplastic
astrocytoma, glioblastoma multiforme), retinoblastoma, pilocytic
astrocytomas (grade I), meningiomas, metastatic brain tumors,
neuroblastoma, pituitary adenomas, skull base meningiomas, and
skull base cancer. As used herein, the term "nervous system tumors"
refers to a condition in which a subject has a malignant
proliferation of nervous system cells.
[0074] Cancers that can be treated by the present monoterpene (or
sesquiterpene) derivatives include, but are not limited to, lung
cancer, ear, nose and throat cancer, leukemia, colon cancer,
melanoma, pancreatic cancer, mammary cancer, prostate cancer,
breast cancer, hematopoietic cancer, ovarian cancer, basal cell
carcinoma, biliary tract cancer; bladder cancer; bone cancer;
breast cancer; cervical cancer; choriocarcinoma; colon and rectum
cancer; connective tissue cancer; cancer of the digestive system;
endometrial cancer; esophageal cancer; eye cancer; cancer of the
head and neck; gastric cancer; intra-epithelial neoplasm; kidney
cancer; larynx cancer; leukemia including acute myeloid leukemia,
acute lymphoid leukemia, chronic myeloid leukemia, chronic lymphoid
leukemia; liver cancer; lymphoma including Hodgkin's and
Non-Hodgkin's lymphoma; myeloma; fibroma, neuroblastoma; oral
cavity cancer (e.g., lip, tongue, mouth, and pharynx); ovarian
cancer; pancreatic cancer; prostate cancer; retinoblastoma;
rhabdomyosarcoma; rectal cancer; renal cancer; cancer of the
respiratory system; sarcoma; skin cancer; stomach cancer;
testicular cancer; thyroid cancer; uterine cancer; cancer of the
urinary system, as well as other carcinomas and sarcomas. U.S. Pat.
No. 7,601,355.
[0075] The present invention also provides methods of treating CNS
disorders, including, without limitation, primary degenerative
neurological disorders such as Alzheimer's, Parkinson's,
psychological disorders, psychosis and depression. Treatment may
consist of the use of a monoterpene or sesquiterpene derivative
alone or in combination with current medications used in the
treatment of Parkinson's, Alzheimer's, or psychological
disorders.
[0076] The present invention also provides a method of improving
immunomodulatory therapy responses comprising the steps of exposing
cells to an effective amount of a monoterpene or sisquiterpene
derivative, such as a POH-TMZ-fatty acid derivative, before or
during immunomodulatory treatment. Preferred immunomodulatory
agents are cytokines, such interleukins, lymphokines, monokines,
interfereons and chemokines.
[0077] The present composition may be administered by any method
known in the art, including, without limitation, intranasal, oral,
transdermal, ocular, intraperitoneal, inhalation, intravenous, ICV,
intracisternal injection or infusion, subcutaneous, implant,
vaginal, sublingual, urethral (e.g., urethral suppository),
subcutaneous, intramuscular, intravenous, rectal, sub-lingual,
mucosal, ophthalmic, spinal, intrathecal, intra-articular,
intra-arterial, sub-arachinoid, bronchial and lymphatic
administration. Topical formulation may be in the form of gel,
ointment, cream, aerosol, etc; intranasal formulation can be
delivered as a spray or in a drop; transdermal formulation may be
administered via a transdermal patch or iontorphoresis; inhalation
formulation can be delivered using a nebulizer or similar device.
Compositions can also take the form of tablets, pills, capsules,
semisolids, powders, sustained release formulations, solutions,
suspensions, elixirs, aerosols, or any other appropriate
compositions.
[0078] To prepare such pharmaceutical compositions, one or more of
monoterpene (or sesquiterpene) derivatives may be mixed with a
pharmaceutical acceptable carrier, adjuvant and/or excipient,
according to conventional pharmaceutical compounding techniques.
Pharmaceutically acceptable carriers that can be used in the
present compositions encompass any of the standard pharmaceutical
carriers, such as a phosphate buffered saline solution, water, and
emulsions, such as an oil/water or water/oil emulsion, and various
types of wetting agents. The compositions can additionally contain
solid pharmaceutical excipients such as starch, cellulose, talc,
glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk,
silica gel, magnesium stearate, sodium stearate, glycerol
monostearate, sodium chloride, dried skim milk and the like. Liquid
and semisolid excipients may be selected from glycerol, propylene
glycol, water, ethanol and various oils, including those of
petroleum, animal, vegetable or synthetic origin, e.g., peanut oil,
soybean oil, mineral oil, sesame oil, etc. Liquid carriers,
particularly for injectable solutions, include water, saline,
aqueous dextrose, and glycols. For examples of carriers,
stabilizers and adjuvants, see Remington's Pharmaceutical Sciences,
edited by E. W. Martin (Mack Publishing Company, 18th ed., 1990).
The compositions also can include stabilizers and
preservatives.
[0079] As used herein, the term "therapeutically effective amount"
is an amount sufficient to treat a specified disorder or disease or
alternatively to obtain a pharmacological response treating a
disorder or disease. Methods of determining the most effective
means and dosage of administration can vary with the composition
used for therapy, the purpose of the therapy, the target cell being
treated, and the subject being treated. Treatment dosages generally
may be titrated to optimize safety and efficacy. Single or multiple
administrations can be carried out with the dose level and pattern
being selected by the treating physician. Suitable dosage
formulations and methods of administering the agents can be readily
determined by those of skill in the art. For example, the
composition are administered at about 0.01 mg/kg to about 200
mg/kg, about 0.1 mg/kg to about 100 mg/kg, or about 0.5 mg/kg to
about 50 mg/kg. When the compounds described herein are
co-administered with another agent or therapy, the effective amount
may be less than when the agent is used alone.
[0080] Transdermal formulations may be prepared by incorporating
the active agent in a thixotropic or gelatinous carrier such as a
cellulosic medium, e.g., methyl cellulose or hydroxyethyl
cellulose, with the resulting formulation then being packed in a
transdermal device adapted to be secured in dermal contact with the
skin of a wearer. If the composition is in the form of a gel, the
composition may be rubbed onto a membrane of the patient, for
example, the skin, preferably intact, clean, and dry skin, of the
shoulder or upper arm and or the upper torso, and maintained
thereon for a period of time sufficient for delivery of the
monoterpene (or sesquiterpene) derivative to the blood serum of the
patient. The composition of the present invention in gel form may
be contained in a tube, a sachet, or a metered pump. Such a tube or
sachet may contain one unit dose, or more than one unit dose, of
the composition. A metered pump may be capable of dispensing one
metered dose of the composition.
[0081] The present invention also provides the compositions as
described above for intranasal administration. As such, the
compositions can further comprise a permeation enhancer. Southall
et al. Developments in Nasal Drug Delivery, 2000. The monoterpene
(or sesquiterpene) derivative may be administered intranasally in a
liquid form such as a solution, an emulsion, a suspension, drops,
or in a solid form such as a powder, gel, or ointment. Devices to
deliver intranasal medications are well known in the art. Nasal
drug delivery can be carried out using devices including, but not
limited to, intranasal inhalers, intranasal spray devices,
atomizers, nasal spray bottles, unit dose containers, pumps,
droppers, squeeze bottles, nebulizers, metered dose inhalers (MDI),
pressurized dose inhalers, insufflators, and bi-directional
devices. The nasal delivery device can be metered to administer an
accurate effective dosage amount to the nasal cavity. The nasal
delivery device can be for single unit delivery or multiple unit
delivery. In a specific example, the ViaNase Electronic Atomizer
from Kurve Technology (Bethell, Washington) can be used in this
invention (http://www.kurvetech.com). The compounds of the present
invention may also be delivered through a tube, a catheter, a
syringe, a packtail, a pledget, a nasal tampon or by submucosal
infusion. U.S. Patent Publication Nos. 20090326275, 20090291894,
20090281522 and 20090317377.
[0082] The monoterpene (or sesquiterpene) derivative can be
formulated as aerosols using standard procedures. The monoterpene
(or sesquiterpene) derivative may be formulated with or without
solvents, and formulated with or without carriers. The formulation
may be a solution, or may be an aqueous emulsion with one or more
surfactants. For example, an aerosol spray may be generated from
pressurized container with a suitable propellant such as,
dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, hydrocarbons, compressed air, nitrogen,
carbon dioxide, or other suitable gas. The dosage unit can be
determined by providing a valve to deliver a metered amount. Pump
spray dispensers can dispense a metered dose or a dose having a
specific particle or droplet size. As used herein, the term
"aerosol" refers to a suspension of fine solid particles or liquid
solution droplets in a gas. Specifically, aerosol includes a
gas-borne suspension of droplets of a monoterpene (or
sesquiterpene), as may be produced in any suitable device, such as
an MDI, a nebulizer, or a mist sprayer. Aerosol also includes a dry
powder composition of the composition of the instant invention
suspended in air or other carrier gas. Gonda (1990) Critical
Reviews in Therapeutic Drug Carrier Systems 6:273-313. Raeburn et
al., (1992) Pharmacol. Toxicol. Methods 27:143-159.
[0083] The monoterpene (or sesquiterpene) derivative may be
delivered to the nasal cavity as a powder in a form such as
microspheres delivered by a nasal insufflator. The monoterpene (or
sesquiterpene) derivative may be absorbed to a solid surface, for
example, a carrier. The powder or microspheres may be administered
in a dry, air-dispensable form. The powder or microspheres may be
stored in a container of the insufflator. Alternatively the powder
or microspheres may be filled into a capsule, such as a gelatin
capsule, or other single dose unit adapted for nasal
administration.
[0084] The pharmaceutical composition can be delivered to the nasal
cavity by direct placement of the composition in the nasal cavity,
for example, in the form of a gel, an ointment, a nasal emulsion, a
lotion, a cream, a nasal tampon, a dropper, or a bioadhesive strip.
In certain embodiments, it can be desirable to prolong the
residence time of the pharmaceutical composition in the nasal
cavity, for example, to enhance absorption. Thus, the
pharmaceutical composition can optionally be formulated with a
bioadhesive polymer, a gum (e.g., xanthan gum), chitosan (e.g.,
highly purified cationic polysaccharide), pectin (or any
carbohydrate that thickens like a gel or emulsifies when applied to
nasal mucosa), a microsphere (e.g., starch, albumin, dextran,
cyclodextrin), gelatin, a liposome, carbamer, polyvinyl alcohol,
alginate, acacia, chitosans and/or cellulose (e.g., methyl or
propyl; hydroxyl or carboxy; carboxymethyl or hydroxylpropyl).
[0085] The composition containing the purified monoterpene (or
sesquiterpene) can be administered by oral inhalation into the
respiratory tract, i.e., the lungs.
[0086] Typical delivery systems for inhalable agents include
nebulizer inhalers, dry powder inhalers (DPI), and metered-dose
inhalers (MDI).
[0087] Nebulizer devices produce a stream of high velocity air that
causes a therapeutic agent in the form of liquid to spray as a
mist. The therapeutic agent is formulated in a liquid form such as
a solution or a suspension of particles of suitable size. In one
embodiment, the particles are micronized. The term "micronized" is
defined as having about 90% or more of the particles with a
diameter of less than about 10 .mu.m. Suitable nebulizer devices
are provided commercially, for example, by PARI GmbH (Starnberg,
Germany). Other nebulizer devices include Respimat (Boehringer
Ingelheim) and those disclosed in, for example, U.S. Pat. Nos.
7,568,480 and 6,123,068, and WO 97/12687. The monoterpenes (or
sesquiterpenes) can be formulated for use in a nebulizer device as
an aqueous solution or as a liquid suspension.
[0088] DPI devices typically administer a therapeutic agent in the
form of a free flowing powder that can be dispersed in a patient's
air-stream during inspiration. DPI devices which use an external
energy source may also be used in the present invention. In order
to achieve a free flowing powder, the therapeutic agent can be
formulated with a suitable excipient (e.g., lactose). A dry powder
formulation can be made, for example, by combining dry lactose
having a particle size between about 1 .mu.m and 100 .mu.m with
micronized particles of the monoterpenes (or sesquiterpenes) and
dry blending. Alternatively, the monoterpene can be formulated
without excipients. The formulation is loaded into a dry powder
dispenser, or into inhalation cartridges or capsules for use with a
dry powder delivery device. Examples of DPI devices provided
commercially include Diskhaler (GlaxoSmithKline, Research Triangle
Park, N.C.) (see, e.g., U.S. Pat. No. 5,035,237); Diskus
(GlaxoSmithKline) (see, e.g., U.S. Pat. No. 6,378,519; Turbuhaler
(AstraZeneca, Wilmington, Del.) (see, e.g., U.S. Pat. No.
4,524,769); and Rotahaler (GlaxoSmithKline) (see, e.g., U.S. Pat.
No. 4,353,365). Further examples of suitable DPI devices are
described in U.S. Pat. Nos. 5,415,162, 5,239,993, and 5,715,810 and
references therein.
[0089] MDI devices typically discharge a measured amount of
therapeutic agent using compressed propellant gas. Formulations for
MDI administration include a solution or suspension of active
ingredient in a liquefied propellant. Examples of propellants
include hydrofluoroalklanes (HFA), such as
1,1,1,2-tetrafluoroethane (HFA 134a) and
1,1,1,2,3,3,3-heptafluoro-n-propane, (HFA 227), and
chlorofluorocarbons, such as CCl.sub.3F. Additional components of
HFA formulations for MDI administration include co-solvents, such
as ethanol, pentane, water; and surfactants, such as sorbitan
trioleate, oleic acid, lecithin, and glycerin. (See, for example,
U.S. Pat. No. 5,225,183, EP 0717987, and WO 92/22286). The
formulation is loaded into an aerosol canister, which forms a
portion of an MDI device. Examples of MDI devices developed
specifically for use with HFA propellants are provided in U.S. Pat.
Nos. 6,006,745 and 6,143,227. For examples of processes of
preparing suitable formulations and devices suitable for inhalation
dosing see U.S. Pat. Nos. 6,268,533, 5,983,956, 5,874,063, and
6,221,398, and WO 99/53901, WO 00/61108, WO 99/55319 and WO
00/30614.
[0090] The monoterpene (or sesquiterpene) derivative may be
encapsulated in liposomes or microcapsules for delivery via
inhalation. A liposome is a vesicle composed of a lipid bilayer
membrane and an aqueous interior. The lipid membrane may be made of
phospholipids, examples of which include phosphatidylcholine such
as lecithin and lysolecithin; acidic phospholipids such as
phosphatidylserine and phosphatidylglycerol; and
sphingophospholipids such as phosphatidylethanolamine and
sphingomyelin. Alternatively, cholesterol may be added. A
microcapsule is a particle coated with a coating material. For
example, the coating material may consist of a mixture of a
film-forming polymer, a hydrophobic plasticizer, a surface
activating agent or/and a lubricant nitrogen-containing polymer.
U.S. Pat. Nos. 6,313,176 and 7,563,768.
[0091] The monoterpene (or sesquiterpene) derivative may also be
used alone or in combination with other chemotherapeutic agents via
topical application for the treatment of localized cancers such as
breast cancer or melanomas. The monoterpene (or sesquiterpene)
derivative may also be used in combination with narcotics or
analgesics for transdermal delivery of pain medication.
[0092] This invention also provides the compositions as described
above for ocular administration. As such, the compositions can
further comprise a permeation enhancer. For ocular administration,
the compositions described herein can be formulated as a solution,
emulsion, suspension, etc. A variety of vehicles suitable for
administering compounds to the eye are known in the art. Specific
non-limiting examples are described in U.S. Pat. Nos. 6,261,547; 6,
197,934; 6,056,950; 5,800,807; 5,776,445; 5,698,219; 5,521,222;
5,403,841; 5,077,033; 4,882,150; and 4,738,851.
[0093] The monoterpene (or sesquiterpene) derivative can be given
alone or in combination with other drugs for the treatment of the
above diseases for a short or prolonged period of time. The present
compositions can be administered to a mammal, preferably a human.
Mammals include, but are not limited to, murines, rats, rabbit,
simians, bovines, ovine, porcine, canines, feline, farm animals,
sport animals, pets, equine, and primates.
[0094] The present invention also provides a method for inhibiting
the growth of a cell in vitro, ex vivo or in vivo, where a cell,
such as a cancer cell, is contacted with an effective amount of the
monoterpene (or sesquiterpene) derivative as described herein.
[0095] Pathological cells or tissue such as hyperproliferative
cells or tissue may be treated by contacting the cells or tissue
with an effective amount of a composition of this invention. The
cells, such as cancer cells, can be primary cancer cells or can be
cultured cells available from tissue banks such as the American
Type Culture Collection (ATCC). The pathological cells can be cells
of a systemic cancer, gliomas, meningiomas, pituitary adenomas, or
a CNS metastasis from a systemic cancer, lung cancer, prostate
cancer, breast cancer, hematopoietic cancer or ovarian cancer. The
cells can be from a vertebrate, preferably a mammal, more
preferably a human. U.S. Patent Publication No. 2004/0087651.
Balassiano et al. (2002) Intern. J. Mol. Med. 10:785-788. Thorne,
et al. (2004) Neuroscience 127:481-496. Fernandes, et al. (2005)
Oncology Reports 13:943-947. Da Fonseca, et al. (2008) Surgical
Neurology 70:259267. Da Fonseca, et al. (2008) Arch. Immunol. Ther.
Exp. 56:267-276. Hashizume, et al. (2008) Neuroncology
10:112-120.
[0096] In vitro efficacy of the present composition can be
determined using methods well known in the art. For example, the
cytoxicity of the present monoterpene (or sesquiterpene) and/or the
therapeutic agents may be studied by MTT
[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide]
cytotoxicity assay. MTT assay is based on the principle of uptake
of MTT, a tetrazolium salt, by metabolically active cells where it
is metabolized into a blue colored formazon product, which can be
read spectrometrically. J. of Immunological Methods 65: 55 63,
1983. The cytoxicity of the present monoterpene (or sesquiterpene)
derivative and/or the therapeutic agents may be studied by colony
formation assay. Functional assays for inhibition of VEGF secretion
and IL-8 secretion may be performed via ELISA. Cell cycle block by
the present monoterpene (or sesquiterpene) derivative and/or the
therapeutic agents may be studied by standard propidium iodide (PI)
staining and flow cytometry. Invasion inhibition may be studied by
Boyden chambers. In this assay a layer of reconstituted basement
membrane, Matrigel, is coated onto chemotaxis filters and acts as a
barrier to the migration of cells in the Boyden chambers. Only
cells with invasive capacity can cross the Matrigel barrier. Other
assays include, but are not limited to cell viability assays,
apoptosis assays, and morphological assays.
[0097] The following are examples of the present invention and are
not to be construed as limiting.
EXAMPLES
Example 1
Preparation of POH-TMZ-linoleate (or TMZ-POH-linoleate, TPL)
triconjugate
((3-Methyl-4-oxo-3,4-dihydro-imidazo[5,1-d][1,2,3,5]tetrazine-8-carbonyl)-
-octadeca-9,12-dienoyl-carbamic acid
4-isopropenyl-cyclohex-1-enylmethyl ester)
[0098] The reaction scheme is as follows:
##STR00047##
[0099] Oxalyl chloride (0.64 g, 5.15 mmol) was added slowly to a
mixture of Temozolamide (Source: OChem Incorporation, Lot
#9110918A; 0.5 g, 2.57 mmol) in 1,2-dichloroethane (10 mL) over a
period of 2 min while maintaining the temperature at 10.degree. C.
under N.sub.2. The reaction mixture was allowed to warm to room
temperature and then heated to reflux for 3 h. The excess of oxalyl
chloride and 1,2-dichloroethane were removed by concentration under
vacuum. The resulting residue was redissolved in 1,2-dichlorethane
(15 mL) and the reaction mixture was cooled to 10.degree. C. under
N.sub.2. A solution of Perillyl alcohol (0.086 g, 0.56 mmol) in
1,2-dichloroethane (3 mL) was added over a period of 5 min. The
reaction mixture was allowed to warm to room temperature and
stirred for 14 h. 1,2-Dichloroethane was concentrated under vacuum
to give a residue which was triturated with hexanes. The resulting
pale-yellow solid (TMZ-POH or POH-TMZ) was filtered and washed with
hexanes. Weight: 0.85 g; Yield: 89%.
[0100] .sup.1H-NMR (400 MHz, CDCl.sub.3): .delta. 1.4-2.2 (m, 10H),
4.06 (s, 3H), 4.6-4.8 (m, 4H), 5.88 (br s, 1H), 8.42 (s, 1H), 9.31
(br s, 1H); MS (APCI): No molecular ion peak is observed. m/e: 314
(100%), 286.5 (17%), 136 (12%).
[0101] POH-TMZ obtained above (300 mg, 0.80 mmol) in dry THF (5.0
mL) was added to Sodium hydride (60%, 48 mg, 1.2 mmol) in dry THF
(4.0 mL) at 0-5.degree. C. The mixture was allowed to warm to
20-25.degree. C. and stirred for 1.0 h. Linoleoyl chloride
(alternatively the acid chloride of any fatty acid or carboxylic
acid, R--COCl, may be used) solution (264 mg, 0.88 mmol) in dry THF
was added slowly over a period of 15 min, while maintaining the
temperature below 10.degree. C. The mixture was slowly heated to
35-40.degree. C. and stirred for 1.0 h. After confirming the
completion of the reaction by TLC (20% EtOAc/Hexanes), the mixture
was quenched with saturated ammonium chloride solution. The
reaction mixture was extracted with ethyl acetate (2.times.15 mL).
The combined organic layer was washed with water (25 mL), brine
(10%, 25 mL) and dried over sodium sulfate. The filtered organic
layer was concentrated to give an oil which was purified by column
chromatography [Column dimensions: dia: 1.5 cm, height: 10 cm,
silica: 230-400 mesh] and eluted with a mixture of 5% ethyl
acetate/hexanes (100 mL) followed by 10% ethyl acetate/hexanes (100
mL). The 10% ethyl acetate/hexanes fractions were combined and
concentrated under vacuum to give a gummy solid. Weight: 260 mg;
Yield: 50%.
Example 2
Cytotoxicity of POH-TMZ-Linoleate (TPL)
[0102] The cytotoxicity of TMZ, POH-TMZ, and POH-TMZ-Linoleate were
studied by colony formation assay of A2058 and A375 melanoma cells.
A2058 and A375 cells were harvested and made single cell suspension
in culture medium (DMEM with 10% FBS, 1% Penicillin/Streptomycin).
500 cells/well were seeded into 12-well plate (tissue culture
treated, from Olympus Plastics) pre-warmed culture medium at
37.degree. C. The cells were cultured overnight for attachment to
the plate. Then the medium was removed and replaced with fresh
medium containing varied concentrations of drugs (or no drugs as a
control) for further 48-hour incubation. After 48 hours treatment,
the medium was aspirated and replaced with normal culture medium.
7-10 days later, the culture medium was aspirated, and the colonies
formed inside the wells were washed with cold PBS once. Then the
colonies were stained with 0.1% methylene blue (in methanol) for
4-6 hours. The plates were then washed and the formed colonies
counted (the colonies formed with 20 cells or more are countable).
The colonies count results shown in FIGS. 1 and 2 indicate that, at
similar concentrations, POH-TMZ-linoleate was more effective in
killing the melanoma cells than POH-TMZ, which is in turn more
effective than TMZ.
Example 3
In Vivo Tumor Growth Inhibition by POH-TMZ-Linoleate (TPL)
[0103] Inhibition of tumor growth by POH-TMZ-linoleate was studied
in nude mice. Nude mice were injected subcutaneously with
2.times.10.sup.6 human melanoma tumor cells, A2058. The melanoma
cells were allowed to form a palpable nodule in the mice. When the
tumor size reached 1.0-1.5 cm in diameter in any dimension, a
control mouse was treated topically with a vehicle (10% DMSO in 45%
Glycerol+45% ethanol), and a test mouse was topically treated with
TPL (50 mM TPL working solution was reconstituted in 45%
Glycerol+45% Ethanol, and 100 .mu.l (25 mg/kg) was applied
topically on the skin which covers the whole tumor). The control
mouse and the test mouse were both treated twice daily for 14 days.
It was observed that the tumor of the test mouse developed a hard
surface after 14 days of treatment (FIG. 3B), with reduced
vascularization compared with the tumor before the treatment (FIG.
3A).
[0104] After the 14 days of treatment the mice were sacrificed, the
tumor removed, sectioned at 8 .mu.m, fixed with acetone, and stored
at -20.degree. C. For histological analysis, the tissues were
blocked with sea block and stained with the primary antibody Rat
anti Mouse (1:50) overnight following which secondary biotinylated
goat anti rat antibody (1:200) was added for 45 minutes, washed and
avidin biotin complex, ABC elite was added for 30 minutes, washed
and then stained with AEC and counter stained with hematoxylin.
Such staining was used to evaluate the endothelial marker CD31
expression, which is representative of the amounts of blood vessels
in the tumor. Staining images were captured at 40.times. under
optical microscope. A visual inspection of the stained tissues
showed that the tumor angiogenesis was much lower in the
TPL-treated mouse (FIGS. 4D-4F) than that in the vehicle-treated
mouse (FIGS. 4A-4C). The histological analysis results were further
quantified using ImageJ software by measuring the average area of
staining in the tumors of the test mouse after TPL treatment and
the control mouse, where the average area of staining was
calculated based on the red precipitate of the stain in
.mu.m.sup.2. From FIG. 5, it can be seen that the average area of
staining is much smaller in the tumor of the test mouse than that
in the control mouse. These comparisons demonstrate the efficacy of
TPL in treating melanoma.
[0105] The scope of the present invention is not limited by what
has been specifically shown and described hereinabove. Variations,
modifications and other implementations of what is described herein
will occur to those of ordinary skill in the art without departing
from the spirit and scope of the invention.
* * * * *
References